CpG oligodeoxynucleotide (CpG ODN) is a short (around 20 base pair), single-stranded synthetic DNA fragment containing the immunostimulatory CpG motif, a potent agonist for Toll-like receptor 9 (TLR9), activates dendritic cells (DCs) and B cells to produce type I interferons (IFNs) and inflammatory cytokines (non-patent documents 1, 2), and acts as an adjuvant towards both Th1-type humoral and cellular immune responses including cytotoxic T-lymphocyte (CTL) responses (non-patent documents 3, 4). Therefore, CpG ODN has been postulated as a possible immunotherapeutic agent against infectious diseases, cancer, asthma, and pollinosis (non-patent documents 2, 5)
There are at least four types of CpG ODN, each of which has a different backbone, sequence, and immunostimulatory properties (non-patent document 6). D (also called A) type CpG ODN, typically comprise one palindromic CpG motif with a phosphodiester (PO) backbone and phosphorothioate (PS) poly G tail, that activates plasmacytoid DCs (pDCs) to produce a large amount of IFN-α, but fails to induce pDC maturation and B cell activation (non-patent documents 7, 8). The three other types of ODN consist of a PS backbone. K (also called B) type CpG ODN contains non-palindromic multiple CpG motifs, and strongly activates B cells to produce IL-6 and pDCs to maturation, but barely produce IFN-α (non-patent documents 8, 9). Recently, C and P type CpG ODN have been developed; these contain one and two palindromic CpG sequence(s), respectively, both of which can activate B cells like K type and pDCs like D type, although C type CpG ODN induces weaker IFN-α production compared with P type CpG ODN (non-patent document 10-12). Many superior K type CpG ODNs are described in patent document 1.
The D and P type CpG ODN have been shown to form high-order structures, Hoogsteen base-pairing to form parallel quadruplex structures called G-tetrads, and Watson-Crick base-pairing between cis- and trans-palindromic portions, respectively, that are required for robust IFN-α production by pDCs (non-patent documents 12-14). Although such higher-order structures appear necessary for localization to early endosomes and signaling via TLR9, they suffer from product polymorphisms, aggregation and precipitation, thereby hampering their clinical applications (non-patent document 15). Therefore, only K and C type CpG ODN are generally available as immunotherapeutic agents and vaccine adjuvants for human use (non-patent document 16 and 17). While K type CpG ODN enhances the immunogenicity of vaccines targeting infectious diseases and cancers in human clinical trials (non-patent documents 6, 16), chemical or physical conjugation between antigen and K type CpG ODN is necessary for the optimal adjuvant effects. These results indicate that these four (K, D, P, and C) types of CpG ODN have advantages and disadvantages, however the development of an ‘all-in-one’ CpG ODN activating both B cells and pDCs without aggregation is yet to be accomplished.
Schizophylan (SPG), a soluble β-1,3-glucan derived from Schizophyllum commune, is a drug approved in Japan as an enhancer of radiotherapy in cervical carcinoma patients, for the last three decades (non-patent document 18). Similarly, lentinan (LNT), a soluble β-1,3-glucan derived from shiitake mushroom, is a medicament approved in 1985, and used in combination with a fluoropyrimidine medicament for inoperable and recurrent gastric cancer patients (non-patent documents 19, 20). β-1,3-Glucan has been shown to form complex with poly deoxyadenylate (dA) as a triple helical structure (non-patent document 21).
Patent documents 2-4 disclose use of a water-soluble complex of β-1,3-glucan including schizophyllan and nucleic acid (gene), as a gene carrier. These documents describe that formation of the complex enhances an antisense action of the gene and a resistance action thereof against nuclease.
Patent document 5 discloses use of polysaccharides having a β-1,3-bond as a carrier (transfection agent) enhances the action of an immunostimulating oligonucleotide having a CpG sequence, wherein a phosphodiester bond is substituted by a phosphorothioate bond or phosphorodithioate bond.
Patent document 6 discloses an immunostimulating complex consisting of an immunostimulating oligonucleotide and (β-1,3-glucan having a long β-1,6-glucosidebond side chain.
The present inventors previously demonstrated that mouse and humanized CpG ODN linked with poly dA having a phosphodiester bond at the 5′ end complexed with SPG enhanced cytokine production and acted as an influenza vaccine adjuvant and a prophylactic or therapeutic agent for Th2 cell related diseases (non-patent documents 22, 23, patent document 7). When poly(dA) was added to the 5′-end of CpG of each of K type and D type to form a complex with SPG, the both showed enhanced activity while maintaining the property of K type and D type. However, it was difficult to achieve high yields of the CpG-SPG complex towards its more efficient and cost-effective preclinical as well as clinical development. Recently, when the poly(dA) having phosphorothioate bond linked to CpG ODN, the efficiency of complex formation was elevated by nearly 100% (non-patent document 24). However, a thorough investigation has not yet been conducted to identify the best humanized CpG sequence and optimization of factors to gain “all-in-one” activities of four types of CpG ODN.
Patent document 8 discloses a production method of an antigen/CpG oligonucleotide/β-1,3-glucan type ternary complex.